Azetidine substituted rosamines useful for staining mitochondria

Abstract

Azetidine substitute rosamines useful for staining mitochondria of formula (I) are disclosed, wherein at least one of Y and Z is a substituted or unsubstituted azetidine group; X is selected from O, S, SO2, Se, NR12, P(O)R12, CR13R14, SiR13R14, Te, and GeR13R14, and there is at least one Q group on the pendant phenyl, the Q group comprising a group selected from halo, maleimidyl, OSO2R13 and epoxide. Also disclosed are methods for staining mitochondria involving incubating a sample in a composition comprising the compound, and analysing mitochondria, involving staining a sample of mitochondria, optionally fixing the cells, illuminating the stained sample using light of an appropriate wavelength to fluoresce the compound, and observing or imaging a magnified image of the sample.

1 . A compound comprising a cationic species of formula (I): 
       
         
           
           
               
               
           
         
         or a solvate, or tautomer thereof; and a counter ion; 
         wherein:
 Y is a substituted or unsubstituted azetidine ring and Z is selected from OR 17  or a substituted or unsubstituted azetidine ring; 
 X is selected from O, S, SO 2 , Se, NR 12 , P(O)R 12 , CR 13 R 14 , SiR 13 R 14 , Te, and GeR 13 R 14 ; 
 R 1 , R 2 , R 3 , R 4 , and R 5  are each selected from H, C 1  to C 8  alkyl, OR 15 , C(O)OR 16 , NHC(O)R 15 , C(O)NHR 15 , halo, and a group of formula Q, wherein at least one of R 1 , R 2 , R 3 , R 4 , and R 5  is a group of formula Q; 
 R v , R w , R x , R y , R 6 , R 7  are each independently selected from H, C 1  to C 8  alkyl and halo; 
 R 16  is selected from C 1  to C 8  alkyl, optionally substituted aryl or optionally substituted heteroaryl; 
 R 17  is selected from H, C 1  to C 8  alkyl, optionally substituted aryl or optionally substituted heteroaryl; and 
 Q is a group comprising L-M A , 
 wherein M A  is selected from —CR 18 R 19 M B , NHC(O)CR 18 R 19 M B , C(O)NHCR 18 R 19 M B , NHC(O)-L-CR 18 R 19 M B , —C(O)NH-L-CR 18 R 19 M B , —O—CR 18 R 19 M B  and —O-L-CR 18 R 19 M B ; 
 each M B  is independently selected from halo, maleimidyl, OSO 2 R 13 , and 
 
       
       
         
           
           
               
               
           
         
         each L is an independently selected divalent linker group, substituted or unsubstituted phenylene or is absent, and
 R 18  and R 19 ; are each independently selected from H and CH 3 ; and 
 R 12 , R 13 , R 14 , and R 15  are each independently selected from H, C 1  to C 8  alkyl, optionally substituted aryl or optionally substituted heteroaryl. 
 
       
     
     
         2 . A compound as claimed in  claim 1 , wherein at least one of Y and Z is a substituted or unsubstituted azetidine group of formula: 
       
         
           
           
               
               
           
         
         wherein R A  and R B  are independently selected from H, C 1  to C 8  alkyl, OR 20 , C(O)OR 20 , NHC(O)R 20 , C(O)NHR 20 , halo, NR 20 R 21 , —CN, —NC, optionally substituted aryl or optionally substituted heteroaryl; wherein R 20  and R 21  are independently selected from H, and C 1  to C 8  alkyl. 
       
     
     
         3 . A compound as claimed in  claim 1 , wherein the cationic species is of formula (II): 
       
         
           
           
               
               
           
         
         wherein R 8  and R 9  are independently selected from H, C 1  to C 8  alkyl, OR 20 , C(O)OR 20 , NHC(O)R 20 , C(O)NHR 20 , halo, NR 20 R 21 , —CN, —NC, optionally substituted aryl or optionally substituted heteroaryl. 
       
     
     
         4 . A compound as claimed in  claim 1 , wherein the counter ion is a biologically compatible counter ion. 
     
     
         5 . A compound as claimed in  claim 1 , wherein the counter ion is selected from halide, carboxylate, oxalate, sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate, tetrafluoroborate, tetraphenylboride, hexafluorophosphate, nitrate and anions of aromatic or aliphatic carboxylic acids. 
     
     
         6 . A method as claimed in  claim 1 , wherein the cationic species is of formula (III): 
       
         
           
           
               
               
           
         
         wherein R 10  and R 11  are independently selected from H, C 1  to C 8  alkyl, OR 20 , C(O)OR 20 , NHC(O)R 20 , C(O)NHR 20 , halo, NR 20 R 21 , —CN, —NC, optionally substituted aryl or optionally substituted heteroaryl. 
       
     
     
         7 . A compound as claimed in  claim 1 , wherein the cationic species is of formula (IV): 
       
         
           
           
               
               
           
         
       
     
     
         8 . A compound as claimed in  claim 1 , wherein at least one of R 1  and R 5  is Q. 
     
     
         9 . A compound as claimed in  claim 1 , wherein R 3  is Q. 
     
     
         10 . A compound as claimed in  claim 1 , wherein at least one of R 2  and R 4  is Q. 
     
     
         11 . A compound as claimed in  claim 1 , wherein L comprises an alkylene chain —(CH 2 ) m —, wherein m is 1 to 6. 
     
     
         12 . A compound as claimed in  claim 1 , wherein M B  is halo. 
     
     
         13 . A compound as claimed in  claim 10 , wherein M B  is chloro. 
     
     
         14 . A compound as claimed in  claim 1 , wherein the compound comprises a cationic species selected from species of formulae: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         15 .- 18 . (canceled) 
     
     
         19 . A method for staining mitochondria, the method comprising:
 providing a sample containing mitochondria, and   incubating the sample in a composition comprising a compound as claimed in  claim 1 .   
     
     
         20 . A method as claimed in  claim 19 , wherein incubating the sample is for a predetermined time in the range 10 mins to 2 hours and at a predetermined temperature in the range 20° C. to 39° C. 
     
     
         21 . A method as claimed in  claim 19 , wherein the sample containing mitochondria comprises a tissue sample. 
     
     
         22 . A method as claimed in  claim 19 , wherein the sample containing mitochondria is a plant, animal or fungal tissue sample, a sample of plant, animal or fungal cells or isolated plant, animal or fungal mitochondria. 
     
     
         23 . A method as claimed in  claim 19 , wherein the sample containing mitochondria comprises a sample containing fixed mitochondria and/or a sample containing mitochondria in fixed cells. 
     
     
         24 . A method as claimed in  claim 19 , wherein the sample containing mitochondria contains substantially no live cells. 
     
     
         25 . A method of analysing mitochondria, the method comprising:
 staining a sample of mitochondria using a compound as claimed in  claim 1 ,   fixing the cells,   illuminating the stained sample using light of an appropriate wavelength to fluoresce the compound, and   observing or imaging a magnified image of the sample.   
     
     
         26 . A method as claimed in  claim 25 , wherein the appropriate wavelength is in the range 400 nm to 800 nm. 
     
     
         27 . A method of detecting a mitochondrial condition, the method comprising staining a sample of mitochondria as claimed in claim  18 . 
     
     
         28 . A method as claimed in  claim 27 , wherein the sample of mitochondria is a plant, animal or fungal tissue sample, a sample of plant, animal or fungal cells or isolated plant, animal or fungal mitochondria. 
     
     
         29 . A compound as claimed in  claim 1 , wherein each L is independently selected from C 1  to C 8  alkylene.